Agilent rna 6000 nano kit for bioanalyzer 2100 system

LV tissue (15 to 30 mg) was homogenized in lysis buffer using “PowerGen 125” homogenizer (Fisher Scientific, Ontario, Canada) for 30 seconds. Total RNA was extracted using Qiagen RNeasy Fibrous Tissue Mini Kit (Qiagen, Ontario, Canada), according to manufacturer’s instructions. Genomic DNA contamination was removed by RNase-free DNase I digestion during the extraction procedure (according to the RNA extraction kit protocol). The quality and quantity of total RNA was assessed using Agilent RNA 6000 Nano Kit for Bioanalyzer 2100 System (Agilent Technologies, Santa Clara, California, USA). RNA integrity numbers (RIN) were higher than 8.8 for all samples. 700 ng of total RNA were used for first strand synthesis of complementary DNA (cDNA) using High Capacity cDNA Reverse Transcription Kit with RNase inhibitor (Applied Biosystems, California, USA) in a final reaction volume of 20 µl according to manufacturer’s instructions.

Left ventricular tissue (200 to 400 mg) was homogenized in Trizol buffer using “PowerGen125” homogenizer (Fisher Scientific, ON, Canada) for approximately 60 seconds. Homogenates were then aliquoted into 5 tubes and one of the aliquot was used to extract total RNA using Qiagen miRNeasy Kit (Qiagen, ON, Canada). The quality and quantity of total RNA was assessed using Agilent RNA 6000 Nano Kit for Bioanalyzer 2100 System (Agilent Technologies, Santa Clara, CA, USA). RNA integrity numbers (RIN) were higher than 8/10 for all samples. One (1) μg of total RNA was used to generate the first strand of complementary DNA (cDNA) using High Capacity cDNA Reverse Transcription Kit with RNase inhibitor (Applied Biosystems, CA, USA) in a final reaction volume of 20 μl according to manufacturer’s instructions.